Biosynthesis of Poly-beta-Hydroxyalkanoates from Pentoses by Pseudomonas pseudoflava

The potential of Pseudomonas pseudoflava to produce poly-beta-hydroxyalkanoates (PHAs) from pentoses was studied. This organism was able to use a hydrolysate from the hemicellulosic fraction of poplar wood as a carbon and energy source for its growth. However, in batch cultures, growth was inhibited completely at hydrolysate concentrations higher than 30% (vol/vol). When P. pseudoflava was grown on the major sugars present in hemicelluloses in batch cultures, poly-beta-hydroxybutyric acid (PHB) accumulated when glucose, xylose, or arabinose was the sole carbon source, with the final PHB content varying from 17% (wt/wt) of the biomass dry weight on arabinose to 22% (wt/wt) of the biomass dry weight on glucose and xylose. Specific growth rates were 0.58 h on glucose, 0.13 h on xylose, and 0.10 h on arabinose, while the specific PHB production rates based on total biomass ranged from 0.02 g g h on arabinose to 0.11 g g h on glucose. PHB weight-average molecular weights were 640,000 on arabinose and 1,100,000 on glucose and xylose. The absolute amount of PHB in the cells decreased markedly when nitrogen limitation was relaxed by feeding ammonium sulfate at the end of the PHB accumulation stage of the arabinose and xylose fermentations. Copolymers of beta-hydroxybutyric and beta-hydroxyvaleric acids were produced when propionic acid was added to shake flasks containing 10 g of glucose liter. The beta-hydroxyvaleric acid monomer content attained a maximum of 45 mol% when the initial propionic acid concentration was 2 g liter.     

Effects of methanol on cell growth and lipid production from mixotrophic cultivation of Chlorella sp.

The marine microalga Chlorella sp. was cultivated under mixotrophic conditions using methanol as an organic carbon source, which may also act to maintain the sterility of the medium for long-term outdoor cultivation. The optimal methanol concentration was determined to be 1% (v/v) for both cell growth and lipid production when supplying 5% CO₂ with 450 μE/m²/sec of continuous illumination. Under these conditions, the maximal cell biomass and total lipid production were 4.2 g dry wt/L and 17.5% (w/w), respectively, compared to 2.2 g dry wt/L and 12.5% (w/w) from autotrophic growth. Cell growth was inhibited at methanol concentrations above 1% (v/v) due to increased toxicity, whereas 1% methanol alone sustained 1.0 g dry wt/L and 4.8% total lipid production. We found that methanol was preferentially consumed during the initial period of cultivation, and carbon dioxide was consumed when the methanol was depleted. A 12:12 h (light:dark) cyclic illumination period produced favorable cell growth (3.6 g dry wt/L). Higher lipid production was observed with cyclic illumination than with continuous illumination (18.6% (w/w) vs 17.5% (w/w)), and better lipid production was also obtained under mixotrophic rather than autotrophic conditions. Interestingly, under mixotrophic conditions with 12:12 (h) cyclic illumination, high proportions of C_16:0, C_18:0, and C_18:1 were observed, which are beneficial for biodiesel production. These results strongly indicate that the carbon source is important for controlling both lipid composition and cell growth under mixotrophic conditions, and they suggest that methanol could be utilized to scale up production to an open pond type system for outdoor cultivation where light illumination changes periodically.     

Growth characteristics of a new methylomonad.

A methylomonad culture was isolated from pond water and examined as a potential source of single-cell protein. A medium containing magnesium sulfate, ammonium hydroxide, sodium phosphate, tap water, and methanol supported the growth of the isolate. Optimal growth conditions in batch cultures for the organism were: temperature, 30 to 33 degrees C; pH 7.1; and phosphate concentration, 0.015 M. The minimum doubling time obtained was 1.6 h. The specific growth rate in batch culture was dependent on the methanol concentration, reaching a maximum around 0.2% (wt/vol). Growth inhibition was apparent above 0.3% (wt/vol), and growth was completely inhibited above 4.6% (wt/vol) methanol. Although the inhibitory effect of formaldehyde on the specific growth rate was much greater than that of formate, the organism utilized formaldehyde, but not formate, as a sole carbon and energy source in batch cultures. The isolate was identified primarily by its inability to utilize any carbon source other than methanol and formaldehyde for growth. Although it is capable of rapid growth on methanol, the organism showed a very weak catalase activity. The amino acid content of the cells compared favorably with the reference levels for the essential amino acids specific by the Food and Agricultural Organization of the United Nations.     

Growth characteristics of a new methylomonad.

A methylomonad culture was isolated from pond water and examined as a potential source of single-cell protein. A medium containing magnesium sulfate, ammonium hydroxide, sodium phosphate, tap water, and methanol supported the growth of the isolate. Optimal growth conditions in batch cultures for the organism were: temperature, 30 to 33 degrees C; pH 7.1; and phosphate concentration, 0.015 M. The minimum doubling time obtained was 1.6 h. The specific growth rate in batch culture was dependent on the methanol concentration, reaching a maximum around 0.2% (wt/vol). Growth inhibition was apparent above 0.3% (wt/vol), and growth was completely inhibited above 4.6% (wt/vol) methanol. Although the inhibitory effect of formaldehyde on the specific growth rate was much greater than that of formate, the organism utilized formaldehyde, but not formate, as a sole carbon and energy source in batch cultures. The isolate was identified primarily by its inability to utilize any carbon source other than methanol and formaldehyde for growth. Although it is capable of rapid growth on methanol, the organism showed a very weak catalase activity. The amino acid content of the cells compared favorably with the reference levels for the essential amino acids specific by the Food and Agricultural Organization of the United Nations.     

Issues in the culture of the extremely thermophilic methanogen, Methanothermus fervidus

Basic issues in the culture of the extremely thermophilic archaeon, Methanothermus fervidus, have been investigated, including culture medium formulation, substrate yield and product yield coefficient, growth rate and stoichiometry, and H(2) uptake kinetics. The pH optimum for growth of this organism was estimated at 6.9. Growth medium buffered with PIPES instead of bicarbonate supported both increased growth rate and maximum biomass concentration. Substitution of titanium(III) citrate for the reducing agent sodium sulfide improved culture performance as well. However, independent adjustment of iron and nickel concentrations from 11 to 111 muM, respectively, and carbon dioxide partial pressure from 5 to 20 psia did not impact the culture of M. fervidus significantly. An elemental balance approach was utilized to aid in design of a defined medium to support growth to a target maximum biomass concentration of at least 1.0 g dry wt/L. The growth of this organism was limited by H(2) availability in this reformulated culture medium. The maximum growth rate and biomass concentration achieved in anaerobic vials with the defined medium was 0.16 h(-1) and 0.74 g dry wt/L, respectively. This maximum biomass concentration was a 72% improvement over that obtained with a literature-based defined medium. The Monod parameter, K(s), with H(2) as limiting substrate, was estimated at 1.1 +/- 0.4 psia (55 +/- 20 muM in the broth), based on a H(2) consumption study. Representative values for the substrate yield, Y(X/CO(2) ), and product yield coefficient, Y(CH(4)/) (X), were determined experimentally to be 1.78 +/- 0.04 g dry wt/mol CO(2), and 0.52 +/- 0.01 mol CH(4)/g dry wt, respectively. A bench-scale fermentation system suitable for the culture of extremely thermophilic anaerobes was designed and constructed and proved effective for the culture of M. fervidus. (c) 1993 Wiley & Sons, Inc.     

Modeling Pichia pastoris growth on methanol and optimizing the production of a recombinant protein, the heavy-chain fragment C of botulinum neurotoxin, serotype A

An unstructured growth model for the recombinant methylotrophic yeast P. pastoris Mut(+) expressing the heavy-chain fragment C of botulinum neurotoxin serotype A [BoNT/A(H(c))], was successfully established in quasi-steady state fed-batch fermentations with varying cell densities. The model describes the relationships between specific growth rate and methanol concentration, and the relationships between specific methanol and ammonium consumption rates and specific growth rate under methanol-limited growth conditions. The maximum specific growth rate (mu) determined from the model was 0.08 h(-1) at a methanol concentration of 3.65 g/L, while the actual maximum mu was 0.0709 h(-1). The maximum specific methanol consumption rate was 0.0682 g/g WCW/h. From the model, growth can be defined as either methanol-limited or methanol-inhibited and is delineated at a methanol concentration of 3.65 g/L. Under inhibited conditions, the observed biomass yield (Y(X/MeOH)) was lower and the maintenance coefficient (m(MeOH)) was higher than compared to limited methanol conditions. The Y(X/MeOH) decreased and m(MeOH) increased with increasing methanol concentration under methanol-inhibited conditions. BoNT/A(H(c)) content in cells (alpha) under inhibited growth was lower than that under limited growth, and decreased with increasing methanol concentration. A maximum alpha of 1.72 mg/g WCW was achieved at a mu of 0.0267 h(-1) and induction time of 12 h.     

Environmental control of metabolic fluxes in thermotolerant methylotrophic Bacillus strains

Recently we have isolated a number of thermotolerant, spore-forming methylotrophic bacilli in pure culture. With a methanol-limited chemostat culture of strain Ts1, incremental increases in the incubation temperature from 45°C to 62.5°C revealed an optimum with respect to growth yield of 52.5°C, and a maximum of 62.5°C. Similar investigations revealed a pH optimum of 7.5 and a broad growth rate optimum with respect to growth yield. The organism displayed a low maintenance energy requirement and high growth yield (attained simultaneosly with high growth rates) during growth on methanol. Under all conditions of methanol limitation, substrate was oxidized solely to biomass and CO₂ and carbon recoveries greater than 90% were manifest. Our data suggested that this resulted from an ability of the organism to precisely adjust its catabolic and anabolic pathways to suit prevailing growth conditions. These results are discussed in relation to previously reported data on thermophiles in both batch and chemostat culture.     

Influence of medium salt strength and nitrogen source on biomass and metabolite accumulation in adventitious root cultures of Pseudostellaria heterophylla

We investigated the effects of medium salt strength and ammonia/nitrate ratio on biomass production and metabolites accumulation of adventitious root. The medium with full-strength Murashige and Skoog (MS) reached the highest growth rate (16.77), and the contents of saponin and polysaccharide reached the peak (i.e., 0.65 and 24.85 %) at 3/4 MS and 1 MS, respectively. In case of ammonia/nitrate ratio, a NH₄ ⁺/NO₃ ^? ratio of 20:40 was optimal for the production of biomass and polysaccharide (23.27 %). In contrast, the content of saponin achieved the optimum (0.74 %) at a NH₄ ⁺/NO₃ ^? ratio of 30:30. In 5-L balloon-type bubble bioreactor (BTBB) cultures, an approximately 23-fold increase in biomass was recorded. The fresh weight (FW) and dry weight (DW) were 72.78 and 6.79 g per bioreactor with the contents of saponin (0.62 %) and polysaccharide (17.32 %), respectively. It indicated potential application to produce adventitious roots of pseudostellaria heterophylla with bioreactors on large scale in commercial.     

Improvement of dry matter digestibility of water hyacinth by solid state fermentation using white rot fungi

Feeding value of water hyacinth biomass colonized by three species of white rot fungi during solid-state fermentation was investigated. All three organisms proved to be efficient degraders and enhanced dry matter digestibility. Loss of organic matter was maximum (23.6+/-0.1% dry wt) after 48 days by P. ostreatus. C. indica showed maximum cellulose degradation (18.5+/-0.1% dry wt) than other two fungi after 48 days of incubation. In all cases, an extensive removal of hemicellulose at the initial growth period and a delayed degradation of lignin were observed. Hemicellulolysis was maximum (46.3+/-0.1% dry wt) by C. indica, but delignification (14.2+/-0.2% dry wt) by P. sajor-caju after 48 days. The amount of reducing sugar in the degraded biomass decreased at early stages, but increased as degradation progressed in all three cases (maximum 1.1+/-0.05% dry wt after 48 days by C. indica). Soluble nitrogen content increased only during 16-32 days of incubation (highest 1.1+/-0.1% dry wt after 32 days by P. sajor-caju). Crude protein of the bioconverted biomass increased gradually up to 32 days but decreased thereafter (maximum 10.3+/-0.1% dry wt after 32 days by P. sajor - caju). Per cent change in in vitro dry matter digestibility of degraded substrates enhanced gradually after 8 days and reached maximum after 32 days but thereafter decreased (highest + 20.4+/-0.3% dry wt by P. sajor-caju). The results demonstrated the efficient degrading capacity of the test fungi and their potential use in conversion of water hyacinth biomass into mycoprotein-rich ruminant feed, more so by P. sajor-caju.     

Optimization of microwave-assisted transesterification of dry algal biomass using response surface methodology

The effect of microwave irradiation on the simultaneous extraction and transesterification (in situ transesterification) of dry algal biomass to biodiesel was investigated. A high degree of oil/lipid extraction from dry algal biomass and an efficient conversion of the oils/lipids to biodiesel were demonstrated in a set of well-designed experimental runs. A response surface methodology (RSM) was used to analyze the influence of the process variables (dry algae to methanol (wt/vol) ratio, catalyst concentration, and reaction time) on the fatty acid methyl ester conversion. Based on the experimental results and RSM analysis, the optimal conditions for this process were determined as: dry algae to methanol (wt/vol) ratio of around 1:12, catalyst concentration about 2 wt.%, and reaction time of 4 min. The algal biodiesel samples were analyzed with GC-MS and thin layer chromatography (TLC) methods. Transmission electron microscopy (TEM) images of the algal biomass samples before and after the extraction/transesterification reaction are also presented.     

Methanol as alternative carbon source for quicker efficient production of the microalgae Chlorella minutissima: Role of the concentration and frequence of administration

Autotrophic cultures of the marine microalgae Chlorella minutissima were performed at 13 000 lux continuous illumination in 1 l chambers fertilised with 0.25 g l⁻¹ F2 medium and different doses of methanol. This was administered in two ways during two parallel experimental series of 10 days: 0.05, 0.1, 0.5, 1.0 and 5.0% methanol (v/v) in one unique dose at the beginning of the culture and 1/10 of these (i.e. 0.005, 0.01, 0.05, 0.1 and 0.5% methanol (v/v)) in daily doses for the 10-day culture period. Low concentrations of methanol induced a faster increase of cell density and dry weight than control, while high concentrations induced symptoms of toxicity. The higher cell densities and quicker growth were observed in the experiments with daily administration of 0.005 and 0.1% (v/v) methanol, while those with one dose presented an initial boosted growth but a final cell density lower than control. The role of methanol as alternative carbon source for microalgae, as well as its possible impact on the quality of biomass production and on the environment, are discussed.     

Chemostat optimization of biomass production of a mixed bacterial culture utilizing methanol

Summary A continuous culture technique was used to optimize the medium composition and growth conditions of a mixed bacterial culture utilizing methanol. The improved medium resulted in satisfactory growth, high-yield coefficients and gave a product containing reduced polysaccharide concentrations. Optimal growth and biomass yields occurred at pH 6.8 a temperature of 37° C and dissolved oxygen at >20% saturation. The maximum growth rate was 0.58 h^–1 and maximum biomass yield 0.48 g g^–1. The protein content of the product ranged between 81%–83%, and nucleic acid content between 10%–12%, increasing with growth rate. The amino acid profile of the mixed culture product met and, in some cases, exceeded the UN Food and Agricultural Organization standard, indicating a good source of feed protein.Offprint requests to: A. S. Abu-Ruwaida     

Characteristics of transformedPanax ginseng C.A. Meyer hairy roots: Growth and nutrient profile

Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, which are established via the infection of ginseng root discs withRhizobium rhizogenes, have been used to construct profiles of both biomass growth and nutrient consumption in flask cultures. In a 250 mL shake flask culture, the maximum biomass was observed on the 59th day of the culture period, at 216.8 g (fresh wt) per liter or 11.4 g (dry wt) per liter. The hairy roots were determined to have a growth rate of 0.355 g-DW/g cells/day during the exponential growth phase and a maximum specific growth rate on day 7. Total ginseng saponin and phenolic compound contents were noted to have increased within the latter portion of the culture period. Linear correlations between increases in biomass weight and nutrient uptake were used to imply the conductivity yield 2.60 g-DW/(L·mS) and carbon yield 0.45 g-DW/(g sugar) in the 250 mL flask cultures. The biomass yield when two different nitrogen sources were used (ammonia and nitrate) was shown to remain approximately constant, at 0.47 g-DW/(l·mM NH₄) and 0.33 g-DW/(L·mM NO₃); it remained at these levels for 16 days with the ammonia, and for 24 days with the nitrate. The biomass yield when a phosphate source was used was also shown to remain approximately constant for 9 days, at 3.17 g-DW/(L·mM PO₄), with an R² of 0.99.     

Denitrification of wastewater containing high nitrate and calcium concentrations

The removal of nitrate from rinse wastewater generated in the stainless steel manufacturing process by denitrification in a sequential batch reactor (SBR) was studied. Two different inocula from wastewater treatment plants were tested. The use of an inoculum previously acclimated to high nitrate concentrations led to complete denitrification in 6h (denitrification rate: 22.8mg NO(3)(-)-N/gVSSh), using methanol as carbon source for a COD/N ratio of 4 and for a content of calcium in the wastewater of 150mg/L. Higher calcium concentrations led to a decrease in the biomass growth rate and in the denitrification rate. The optimum COD/N ratio was found to be 3.4, achieving 98% nitrate removal in 7h at a maximum rate of 30.4mg NO(3)(-)-N/gVSSh and very low residual COD in the effluent.     

Standing stock and production of Ecklonia radiata (C.Ag.): J. Agardh

The monthly productivity, standing stock, plant size and density of Ecklonia radiata (C.Ag.) J. Agardh is presented for a 2-yr period. Annual production was 20.7 kg wet wt · m^?2 with maximum growth of 0.9% per day in spring (October–December) and minimum growth of 0.2% per day in late summer. (March–April). A close negative correlation was found between spring and summer growth and water temperature. Maximum biomass (18 kg wet wt · m ^?2) did not coincide with maximum growth but occurred in late summer. Minimum biomass (6 kg wet wt · m ^?2) occurred in winter. An estimate of erosion of plant material from the kelp bed was made from these data and a hypothesis concerning the ultimate destination of eroded and removed kelp plants was formulated.     

Growth characteristics of Saccharomyces rouxii isolated from chocolate syrup.

We investigated the growth parameters of Saccharomyces rouxii isolated from spoiled chocolate syrup. The optimum pH range for S. rouxii was 3.5 to 5.5, whereas the minimum and maximum pH values that permitted growth were 1.5 and 10.5, respectively. For cells grown in 0 and 60% sucrose the optimum water activity (aw) values were 0.97 and 0.96, respectively. The optimum temperature for S. rouxii increased with a decreasing aw regardless of whether glucose or sucrose was used as the humectant. The optimum temperatures for S. rouxii were 28 degrees C at an aw of greater than 0.995 and 35 degrees C at an aw of 0.96 to 0.90 in 2 X potato dextrose broth with sucrose. Increasing the sorbate concentration (from 0.03 to 0.10%) caused the growth of S. rouxii to become more inhibited between aws of greater than 0.995 and 0.82. S. rouxii did not grow when the sorbate level was 0.12% (wt/vol). At lower sorbate levels, the effect of sorbate on the growth of S. rouxii depended on the aw level. Lowering the aw enhanced the resistance of S. rouxii to increasing concentrations of potassium sorbate. Permeability and polyol production are discussed with respect to sorbate tolerance of S. rouxii at different aw levels.     

Quantitative response of cell growth and polysaccharide biosynthesis by the medicinal mushroom Phellinus linteus to NaCl in the medium

The effect of NaCl on cell growth and polysaccharide biosynthesis in the medicinal mushroom Phellinus linteus was studied. With the increase of NaCl concentration between 1 g/l and 7 g/l in the culture medium, the cell growth and intracellular polysaccharide (IPS) accumulation were decreased; extracellular polysaccharide (EPS) concentration was enhanced, with an increase of NaCl concentration from 1 g/l to 3 g/l. Under the optimum NaCl concentration of 3 g/l, the maximum EPS and IPS production reached 2.2±0.15 g/l and 53.6±2.45 mg/g DW on day 12, which improved 32.27% and decreased 16.89% compared to the control, respectively. Both EPS and IPS showed new polysaccharide components by fractionation with DEAE-cellulose ion exchange chromatography compared to the control. The results presented in this study are considered helpful for further investigation on the diversity of polysaccharide biosynthesis of this medicinal fungus under NaCl environments.     

Accumulation of sesquiterpenes and polysaccharides in cells of zedoary (Curcuma zedoaria Roscoe) cultured in a 10 L bioreactor

We developed a cell suspension culture system for zedoary (Curcuma zedoaria Roscoe), using 100 g fresh weight inoculum in a batch culture. The maximum cell biomass of 68.46 g/L fresh weight was obtained after 14 days of culture in a 10 L bioreactor with a pitch-blade impeller maintained at an agitation speed of 150 rpm and an aeration rate of 2.5 L/min. The accumulation of sesquiterpenes and polysaccharide in zedoary cells from 2 to 18 days was measured by HPLC and a phenol-sulfuric acid assay, respectively. The total polysaccharide concentration increased between 2 to 10 days of culture and reached a maximum value of 6.55%. HPLC revealed several eluted peaks of sesquiterpenes, which increased in amplitude from days 2 to 10. Furthermore, our results indicated that biotransformation occurred in the cell suspension, transforming certain sesquiterpenes into other types during culture.     

Growth and fatty acid composition of Nannochloropsis sp. grown mixotrophically in fed-batch culture

The cell growth and eicosapentaenoic acid (EPA) yields of Nannochloropsis sp. were enhanced in the fed-batch cultures. With feeding glucose solution, the biomass reached 1.1 g dry wt l(-1) after 10 days' culture, which was 40% higher than that obtained in the batch culture (0.8 g dry wt l(-1)). With supplement of nitrate solution, the biomass reached 1 g dry wt l(-1), and reached the stationary phase 2 days earlier than the others. The maximum of biomass (1.2 g dry wt l(-1)) was obtained with the supplement of the mixture of glucose and nitrate solution. The EPA yields of Nannochloropsis sp. after 10 days' growth in the fed-batch cultures were 52 mg l(-1), 43 mg l(-1) and 56 mg l(-1) with, respectively, addition of nitrate, glucose and both together. In batch culture only 35 mg EPA l(-1) was obtained.     

Polysaccharide reserve material in the acetotrophic methanogen,Methanosarcina thermophila strain TM-1: accumulation and mobilization

The ability of Methanosarcina thermophila strain TM-1 to store a reserve polysaccharide was studied using both biochemical methods and thin-section electron microscopy. When grown under conditions of excess carbon and energy (either methanol or acetate) and limiting nitrogen, M. thermophila accumulated a polysaccharide which could be hydrolyzed to glucose by the enzyme amyloglucosidase. This polysaccharide reached levels of 20 mg polysaccharide per g protein in nitrogen-limited cells, while cells limited for carbon, as well as cells in the exponential phase of growth, did not accumulate significant amounts of this polysaccharide. Thin-section electron micrographs of M. thermophila showed glycogen-like inclusion granules in nitrogen-limited cells but not in carbon-limited or exponential-phase cells. These granules were stained by a polysaccharide-specific staining procedure, the PATO stain. The polysaccharide was purified from cell extracts, the iodine-polysaccharide complex gave a maximum absorption at between 500 and 510 nm. The polysaccharide was mobilized within 21 h by cells starved for a carbon/energy source. N-Limited (polysaccharide-containing) acetategrown cells could shift to methanogenesis from methanol more quickly than did C-limited acetate-grown cells lacking polysaccharide, and ATP levels remained higher in N-limited cells. The results are consistant with the hypothesis that this polysaccharide can provide carbon and energy for metabolic shifts but other storage compounds, such as polyphosphate, may also play a similar role.